Metagenomic Analysis of Human Diarrhea- Viral detection and discovery
Introduction Human diarrhea can be chronic or acute, depending on the duration. Diarrhea is an increase in the frequency of bowel movements, an increase in the looseness of stool or both. Bacteria, protozoa and viruses have all been linked as a potential causative agents of human diarrhea. School age children are more susceptible to diarrhea, because the often share their lunch without washing their hands. According to a research article published in PLOS pathogen, diarrhea is the primary cause of death in young children. Approximately 1.8 million children worldwide die from diarrhea annually and other cases of diarrhea in millions others suffer multiple episodes of nonfatal diarrhea. There is a possibility that microorganism (e.g.virus) that an unknown microorganism could be responsible for different episodes of human diarrhea and that are where inception of metagenomic approach enables the detection of unexpected virus (es). Metagenomic approaches, such as mass sequencing allows for comprehensive insights into functional diversity of complex microbial population present in stool during episodes of human diarrhea.This study uses a metagenomic approach called “micro-mass sequencing” to systematically identify viruses present in stool from 12 different pediatric patients suffering from acute diarrhea. “This analysis provides evidence for the detection of known enteric viruses, viral co-infections, and novel viruses” (Finkbeiner S., Allred A.,et. al 2008). Material & Method Cohort study of stool samples from tow different hospitals (Melbourne and Seattle) was collected from children with acute diarrhea. Stool samples collected from children under the age of 5 acute diarrheas between 1978 and 1999 (Royal Children's Hospital, Melbourne, Victoria, Australia). * Enzyme immunoassays (EIA) are used to test specimens and culture assays for rotaviruses, adenoviruses, and common bacterial and parasitic pathogens. * RT-PCR assays were used to screen specimens for the presence of caliciviruses and astroviruses Stool samples collected from children under the age of 5 acute diarrheas 2003–2005 (Children’s Hospital and Regional Medical Center in Seattle, Washington, USA). ' * Standard culture assays, Clostridium difficile toxin by a cytotoxicity assay, parasites by microscopy and antigen testing were used to test the specimens for the presence of Campylobacter jejuni, Escherichia coli O157:H7 and non-O157:H7 Shiga toxin-producing E. coli, Salmonella, Shigella, and Yersinia. * The specimen was tested further using EIA for the presence of rotaviruses, adenoviruses, noroviruses 1 & 2, and astroviruses. Result 'Aggregate library analysis Metagenomic analysis of 12 fecal samples from pediatric patients suffering from acute diarrhea: 384 clones were sequenced for each sample library. * A total of 4,608 sequences were generated * 3,169 passed through a quality filter and * 2,013 contained unique sequence information * 1,457 (72%) were identifiable by similarity to sequences in the Genbank nr database based on tBLASTx (E-value ≤10−5) alignments; while the remaining 556 (28%) sequences are not and were tagged of ‘unknown’ origin. Based on their proposed origin, the 1,457 (72%) identifiable sequences were classified further into categories: * 519 (35.6%) were most similar to eukaryotic viruses * 25 (1.7%) to phage, * 857 (58.8%) to bacteria * 3 (0.2%) to fungi * 20 (1.4%) to human sequences * 33 (2.3%) to other (the sequences had significant hits to mouse, fish, and plant genomes). Individual library statistics For each individual sample, 384 clones were sequenced * proportion of high quality sequences for each sample range from 40% and 95% of the total clones * Percentages of unique sequences per sample ranged from 41% to 97% of the high quality read, with an average length rang from 255 to 626 bp. * Viral sequences constituted between 0–100% of the reads in each library Other libraries * D01 and D05 composed predominantly of viral sequences (64% and 95% respectively) * D08 and D12 consisted largely of bacterial sequences * D03 and D07 consisted of unassigned sequences. Sequences with similarity to viruses from 7 different viral families and three unclassified genera (picobirnavirus, anellovirus and mimivirus) were detected in the 12 different samples, based on the initial BLAST classification criteria. “Five of the samples (D03, D05, D06, D08, and D12) contained sequences from at least two different virus families known to infect humans.” Conclusion Nine putatively novel viruses were detected from sequences of known diarrhea-causing viruses such as picobirnavirus, enterovirus, noroviruses, astroviruses, and Nodaviruses etc. Sequences from a number of novel viruses were detected, some that differed quite significantly from any previously described virus. This study is inconclusive and requires further studies to specifically address the potential of these viruses to cause human disease. References Fierer, N., Breitbart, M., Nulton, J., Salamon, P., Lozupone, C., Jones, R., Robeson,M., Edwards, R.A., Felts, B., Rayhawk, S., et al, 2007.Metagenomic and small-subunit rRNA analyses reveal the genetic diversity of bacteria, archaea, fungi, and viruses in soil. Applied and Environmental Microbiology 73, 7059-7066. Finkbeiner SR, Allred AF, Tarr PI, Klein EJ, Kirkwood CD, et al. (2008) Metagenomic Analysis of Human Diarrhea: Viral Detection and Discovery. PLoS Pathog 4(2): e1000011. doi:10.1371/journal.ppat.1000011 Zhang, T., M. Breitbart, W. H. Lee, J. Q. Run, C. L. Wei, S. W. Soh, M. L. Hibberd, E. T. Liu, F. Rohwer, and Y. Ruan. 2006. RNA viral community in human feces: prevalence of plant pathogenic viruses. PLoS Biol.4:e3